1. Field of the Invention
The present invention relates generally to the fields of virology, molecular biology and medicine. More particularly, it concerns methods involving attenuated reoviruses for screening or selectively killing certain (e.g., cancerous) sub-populations of cells in vitro or from a larger mixed cell population.
2. Description of Related Art
Reovirus (Respiratory Enteric Orphan virus) is a ubiquitous, non-enveloped virus containing 10 segments of double-stranded RNA as its genome, with human infections that are generally mild, restricted to the upper respiratory and gastrointestinal tracts and often asymptomatic, in immune functional hosts (Tyler, 2001). Attempts to reverse engineer reoviruses have been largely unsuccessful due to several factors, including the double-stranded RNA genome of reoviruses. Reoviral particles lacking σ1 have been understood to be non-infectious (Larson et al., 1994).
Importantly, reovirus has been recognized for many years as displaying striking cytocidal activity when it infects certain types of transformed cells (Duncan et al., 1978; Hashiro et al., 1977). Replication-competent oncolytic viruses provide an attractive anti-cancer therapeutic approach. These oncolytic viruses have two principal advantages. Firstly, unlike conventional chemotherapy and radiotherapy, they specifically target cancer cells because of their restricted ability to replicate in normal cells. Secondly, as compared to replication-incompetent vectors, they can propagate from initially infected cancer cells to surrounding cancer cells, thereby achieving a large volume of distribution and potent anti-cancer effects.
Exposure to wild-type (wt) reoviruses, although often asymptomatic in healthy individuals, can nonetheless present a substantial and potentially very serious risk to immunocompromised individuals, limiting the clinical potential of reoviral therapies in patients such as cancer patients who might otherwise benefit from such a therapy. The underlying basis for reoviral oncolytic activity remained unknown until it was shown that transformed cells containing oncogenic Ras-signaling pathways were preferentially susceptible to reovirus infection (type 3 Dearing strain) in vitro and in vivo (Coffey et al., 1998; Strong et al., 1998; Norman et al., 2004). As Ras gene mutations are frequently observed in various types of human cancers (Duursma et al., 2003), these findings have led to the current use of reoviral therapy in clinical trials (Norman et al., 2005). In immune compromised hosts such as newborn and SCID (severe combined immunodeficiency) animals, however, the wt reovirus exerts significant viral pathogenesis especially in neural tissue and cardiac muscle tissue (Sabin, 1959; Weiner et al., 1977; Baty et al., 1993; Loken et al., 2004). In some cases, even in immune-competent hosts including humans, wt reovirus has been associated with viral pathogenesis (Terheggen et al., 2003, Hirasawa et al., 2003). Therefore, especially in immune-compromised or very young hosts, wt reovirus does not always act in a benign manner. For example, in immunocompromised hosts such as very young or immunodeficient adult animals, this virus also has been shown to infect some healthy tissues such as heart, liver, pancreas and neural structures. This concern may further apply to cancer patients treated with extensive radio/chemotherapy as they can be subject to immunosuppression. Thus, there is a clear need to develop a less virulent reovirus for viral oncolytic therapy.
Further, wt reoviruses in many instances possess undesirable virulence and infectivity which limit their potential use in vitro. Specifically, exposure of a cell culture (e.g., a bone marrow transplant taken from a cancer patient) to wt reoviruses may result in the undesirable side effect of killing cells such as stem cells which would be needed for a purpose such as repopulating the immune system of a cancer patient. Thus, the increased virulence of WT reoviruses present significant limitations for the clinical potential of in vitro applications involving exposure of cells to reoviruses. Certain methods relating to the exposure of cellular populations to wt reoviruses have been described (e.g., U.S. Pat. No. 6,994,858, U.S. Patent Publication 2004/0109878, U.S. Patent Publication 2002/0037543, U.S. Patent Publication 2006/0029598, U.S. Patent Publication 2005/0026289, U.S. Patent Publication 2002/0006398, U.S. Patent Publication 2001/0048919 which are incorporated by reference in their entirety without disclaimer); however these methods do not resolve the above-mentioned limitations involving the use of WT reoviruses. Clearly, there exists a need for improved methods for selective killing or purging of cellular sub-populations (e.g., cancerous cells) from a mixed cellular population while limiting damage to necessary or beneficial cells (e.g., stem cells, hematopoietic stem cells).